All proliferating eucaryotic cells must undergo a process termed mitosis before separating into two new cells. Mitosis is a process in which the parent or replicating cell undergoes a series of molecular events that results in the formation of two nuclei in the place of one. Traditionally, mitosis has been described as a series of six dynamic stages: prophase, prometaphase, metaphase, anaphase, telophase and cytokinesis. Briefly, mitosis begins in prophase with the formation of the mitotic spindle having two centrosomes and associated microtubules. During prometaphase and metaphase the centrosomes migrate to opposite ends of the cell to form two spindle poles, followed by the previously duplicated chromosomes aligning at the metaphase plate in-between the two poles. One of each duplicated chromosomes is separated to each pole, nuclei are re-form containing the complement of chromosomes, and the cytoplasm cleaved in half to form two separate daughter cells.
Critical to the separation of the replicated chromosomes and formation of the two nuclei in the mitotic process is the bipolar mitotic spindle. Cells must properly form a bipolar mitotic spindle with bivalent chromosomes properly attached to each pole of the spindle (Gorbsky et al, Bioessays, 19: 193-197, 1997; Hardwick, K. G., Trends Genet., 14: 1-4, 1998). Cells which do not form a correct mitotic spindle arrest at metaphase of mitosis indefinitely or progress into apoptosis. Several proteins from yeast and mammals have been implicated in this process; MAD1 (mitotic arrest deficient), MAD2, and MAD3 (Li et al, Cell, 66: 519-531, 1991 (published erratum appears in Cell, 79(2), following p388)), BUB1 (budding uninhibited by benzimidazole), BUB2 and BUB3 (Hoyt et al, Cell, 66: 507-517, 1991). Mammalian counterparts for these proteins include HsMAD2 (Li et al, Supra) and hBUB1 (Cahill et al, Nature, 392: 300-303, 1998).
One of the most crucial and tightly regulated events during mitosis is centrosome duplication (Schatten, G., Dev. Biol., 165: 299-335, 1994; Balczon, R., International Review of Cytology, 169: 25-82, 1996). The centrosome is an organelle consisting of a pair of centrioles surrounded by an amorphous electron dense material and represents the mammalian equivalent of the yeast spindle pole body. This organelle serves as a site of microtubule organization in the cell. During cell cycle progression the centrosome duplicates, separates and functions as the poles for the mitotic spindle. It is crucial for proper chromosome segregation and fidelity that centrosome replication be tightly regulated, doubling just once during each cell cycle. Centrosome regulation is tightly linked to the S-phase checkpoint (Khan et al., Cancer Res., 58: 396-401, 1998; Lanni et al., Mol. Cell Biol., 18: 1055-1064, 1998). For example blocking cells at the beginning of S-phase leads to the formation of multiple centrosomes (Baczon et al., J. Cell Biol., 130: 105-115, 1995).
Considerable efforts are underway to develop new anti-proliferative agents for use as therapies in the treatment of cancer, as well as non-cancer proliferative disorders such as epithelial hyperplasia, polycytemia, erythrocytemia, thrombocytemia, EBV transformed lymphoproliferative syndrome, dysplastic nevus syndrome, restenosis after angioplasty for coronary heart disease, mastocytosis, histiocytosis, psoriasis, polyps, and the like. One target for anti-proliferative agents is the mitotic pathway. Accordingly, there is a need for the development of novel, effective anti-proliferative agents that target the mitotic pathway.
Vanadocene dichloride (VDC) has been shown to arrest tumor cell growth (Kopf-Maier, et al, J. Cancer Res. Clin. Onccol., 106: 44-52. 1983), and the oxovanadium compound, [VO(Phen)(H2O)2](SO4), has been shown to be an active agent against pharyngonasal cancer as determined by a single assay (Sakurai, et. al, BBRC, 206; 133, 1995). Vanadium compounds have also been shown to induce apoptosis in certain cancer cells (Uckun et al., WO 00/35930).
Against this backdrop the present invention has been developed.
It has now been found that vanadium compounds of the invention, and particularly vanadium compounds and oxovanadium compounds described herein, are effective anti-proliferation agents. These compounds act to disrupt mitotic and meiotic spindle formation and thus are useful to prevent cell mitosis (proliferation) and meiosis.
The invention provides a method for disrupting mitosis or meiosis comprising administering to a subject a effective mitosis or meiosis disrupting amount of a vanadium compound, preferably a vanadium cyclopentadienyl compound (vanadocene), or an oxovanadium compound. Exemplary compounds useful in the method of the invention are described, for example, in published PCT applications W099/36063; WO 00/27389; and WO 00/35930. VDC and VDacac are specific compounds useful in the method invention, as well as other compounds of the invention described below.
The invention are useful applications where disruption of meiosis or mitosis is advantageous, for example, the treatment and prevention of cancer and non-cancer proliferative disorders including those described above, as well as in any other applications where the inhibition of mitosis and/or meiosis in cells is desired or useful.
The present invention is drawn to the use of vanadium compounds, preferably vanadium cyclopentadienyl compounds (vanadocenes) and oxovanadium compounds, including, but not limited to those described in published PCT applications WO99/36063; WO 00/27389; and WO 00/35930. Vanadium compounds useful in the method invention include vanadocene compounds such as vanadocene dichloride (VDC), vandocene acetylacetonate (VDacac), and those vanadium compounds shown below. Specifically, the present invention relates to the finding that these compounds effect disruption of normal mitotic and meiotic spindle formation, and are inhibitors of mitosis and meiosis. The anti-mitotic and anti-meiotic activity makes these compounds particularly attractive anti-proliferative agents, particularly for the treatment of non-cancer proliferative disorders.
Vanadium is a physiologically essential element that can be found in both anionic and cationic forms with oxidation states ranging from xe2x88x923 to +5 (Ixe2x80x94V). This versatility provides unique properties to vanadium complexes. In particular, the catonic form of vanadium complexes having an oxidation state of +4 (IV) has been shown to function as a modulator of cellular redox potential, to regulate enzymatic phosphorylation, and to exert pleiotropic effects in multiple biological systems by catalyzing the generation of reactive oxygen species (ROS). Besides the ability of vanadium metal to assume various oxidation states, its coordination chemistry also plays a key role in its interactions with various biomolecules. In particular, it is demonstrated herein that vanadium compounds, such as vanadium cyclopentadienyl compounds, or derivatives thereof, exhibit anti-mitotic and anti-meiotic properties. The effects of vandocene are primarily via disruption of mitotic and meiotic spindle formation.
The following terms and phrases as used herein have the noted definitions, unless otherwise described:
xe2x80x9cHaloxe2x80x9d is fluoro, chloro, bromo, or iodo.
xe2x80x9cAlkylxe2x80x9d, xe2x80x9calkoxyxe2x80x9d, etc. denote both straight and branched hydrocarbon groups; but reference to an individual radical such as xe2x80x9cpropylxe2x80x9d embraces only the straight chain radical, a branched chain isomer such as xe2x80x9cisopropylxe2x80x9d is specifically referenced.
xe2x80x9cOrganometallic compoundxe2x80x9d is an organic compound comprised of a metal attached directly to carbon (Rxe2x80x94M).
xe2x80x9cCoordination compoundxe2x80x9d is a compound formed by the union of a central metal atom or ion with ions or molecules called ligands or complexing agents.
xe2x80x9cLigandxe2x80x9d or a xe2x80x9ccomplexing agentxe2x80x9d is a molecule, ion or atom that is attached to the central metal atom or ion of a coordination compound.
xe2x80x9cMonodentate ligandxe2x80x9d is a ligand having a single donor atom coordinated to the central metal atom or ion.
xe2x80x9cBidentate ligandxe2x80x9d is a ligand having two donor atoms coordinated to the same central metal atom or ion.
xe2x80x9cVanadocenexe2x80x9d is a compound having a central vanadium metal ion coordinated with at least two cyclopentadiene groups.
xe2x80x9cNon-cancer proliferative disorderxe2x80x9d includes such disorders as non-cancer proliferative disorders such as epithelial hyperplasia, polycytemia, erythrocytemia, thrombocytemia, EBV transformed lymphoproliferative syndrome, dysplastic nevus syndrome, restenosis after angioplasty for coronary heart disease, mastocytosis, histiocytosis, psoriasis, polyps, and the like.
It will be appreciated that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein. Methods to prepare optically active forms are known, for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase. Methods for determining anti-mitotic and anti-meiotic activity of a compound are known, for example, using the standard tests described herein, or other known tests.
Specific values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.
For example, (C1-C6) alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C1-C3) alkyl can be methyl, ethyl or propyl; halo(C1-C3) alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; (C1-C3) alkoxy can be methoxy, ethoxy, or propoxy; and (C2-C6) alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.
The following glossary of vanadium compounds is provided to clarify terms used throughout the specification and provides a listing of exemplary vanadium compounds useful in the method invention:
Unless otherwise indicated, the following abbreviations are used throughout the remainder of the disclosure:
Cp, cyclopentadienyl
Cpxe2x88x92, cyclopentadienyl anion
acac, acetonylacetonate
Bpy, 2,2xe2x80x2 Bipyridine
Hfacac, hexafluoroacetylacetonate
Cat, catecholate
Dtc, diethyl dithio carbamate
Phen, phenanthroline
PH, N-phenyl benzohydroxamic acids
H, acetohydroxamic acid
OTf, trifluoromethane sulphonate
THF, tetrahydrofuran
DMSO, dimethyl sulfoxide
CH3CN, acetonitrile
CH2Cl2, dichloromethane
d-d, laportte spin forbidden transitions
LMCT, ligand to metal charge transfer transitions
p-p*, intraligand charge transfer transitions
The present invention concerns vanadium compounds, and the finding that such compounds have potent and selective anti-mitotic activity, and are particularly active and stable agents for use in the treatment or inhibition of proliferative type cellular disorders, for example, cancer, pathologic hyperplasia, etc.
The vanadium compounds of the invention are also useful for disrupting or inhibiting meiosis, where disruption of meiosis is desired or useful
Vanadium (IV) compounds for use in this invention are as shown in formula I and formula II: 
where R1 and R2 are each independently a monodentate ligand or together form a bidentate ligand; R3 and R4 are each independently a monodentate ligand or together form a bidentate ligand; and R5 is a monodentate ligand, or is absent.
Suitable monodentate ligands include monodentate ligands are selected from the group consisting of halo, OH2, O3SCF3, N3, CN, OCN, SCN, SeCN, and a cyclopentadienyl ring, wherein each cyclopentadienyl ring is optionally substituted with one or more (C1-C3)alkyl. Suitable bidentate ligands are selected from the group consisting of acac, Bpy, Hfacac, Cat, Dtc, PH, H, and Phen, or derivatives thereof. The bidentate ligands may be substituted, for example, with one or more (C1-C3) alkyl, halo, (C1-C3) alkoxy, and halo (C1-C3) alkyl, and derivatives thereof. Halo is chloro, bromo, or iodo, and preferably is chloro.
In one embodiment, a useful vanadium compound has the following structure: 
where R1 and R2 are each independently a monodentate ligand or together form a bidentate ligand; and R3 and R4 are each independently a cyclopentadienyl ring, wherein each cyclopentadienyl ring is optionally substituted with one or more (C1-C3)alkyl. In some preferred embodiments, R1 and R2 are each independently a monodentate ligand selected from the group consisting of of halo, OH2, O3SCF3, N3, CN, OCN, SCN, SeCN, and a cyclopentadienyl ring, where each cyclopentadienyl ring is optionally substituted with one or more (C1-C3)alkyl. Preferably, R1 and R2 arehalo, and more preferably are chloro.
In some other embodiments, R1 and R2 together form a bidentate ligand selected from the group consisting of acac, Bpy, Hfacac, Cat, Dtc, PH, H, and Phen, or derivatives thereof. Preferably, the bidentate ligand is acac, or derivatives thereof.
Some specific examples of compounds of formula I are: VCp2Cl2 (VDC), VCp2Br2, VCp2I2, VCp2(N3)2, VCp2(CN)2, VCp2(NCO)2, VCp2(NCO)Cl, VCp2(NCS)2, VCp2(NCSe)2, VCp2Cl(CH3CN)(FeCl4), VCp2(O3SCF3)2, V(MeCP)2Cl2, V(Me5Cp)2Cl2, VCp2(acac) (VDacac), VCp2(hf-acac), VCp2(bpy), VCp2(cat), VCp2(dtc), VCp2PH, or VCp2H. Two particularly useful vandocene compounds are VDC and VDacac.
Useful oxovanadium compounds of formula II include the compound has the following structure: 
where R1 and R2 are each independently a monodentate ligand or together form a bidentate ligand; R3 and R4 together form a bidentate ligand; and R5 is a monodentate ligand, or is absent. Preferably, R1 and R2 are each independently a monodentate ligand selected from the group consisting of halo, OH2, O3SCF3, N3, CN, OCN, SCN, SeCN, and a cyclopentadienyl ring, wherein each cyclopentadienyl ring is optionally substituted with one or more (C1-C3)alkyl, and R3 and R4 together form a bidentate ligand selected from the group consisting of acac, Bpy, Hfacac, Cat, Dtc, PH, H, and Phen, or derivatives thereof. Where R1 and R2 together form a bidentate ligand, the bidentate ligand is selected from the group consisting of acac, Bpy, Hfacac, Cat, Dtc, PH, H, and Phen, or a derrivative thereof.
Specific compounds of formula II include [VO(phen)], [VO(phen)2], [VO(Me2-phen) ], [VO(Me2-phen)2], [VO(Cl-phen)], [VO(Cl-phen)2], [VO(bipy)], [VO(bipy)2], [VO(Me2-bipy)], [VO(Me2-bipy)2], and [VO(Br,OH-acph)2].
Compositions comprising these vanadium compounds are useful to inhibit mitosis and miosis, and thus are useful in the treatment of numerous proliferative cellular disorders in animals, and in particular mammals. Administration of the compounds as salts may be appropriate. Examples of acceptable salts include alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts, however, any salt that is non-toxic and effective when administered to the animal being treated is acceptable.
Acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently acidic compound with a suitable base affording a physiologically acceptable anion.
The compositions of the invention can be formulated as pharmaceutical compositions and administered to an animal host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient""s diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained. When administered orally, the compositions of the invention can preferably be administered in a gelatin capsule.
The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
The compositions of the invention may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active composition can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active composition in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the present compositions may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
Examples of useful dermatological compositions which can be used to deliver the compounds of formula I to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
Useful dosages of the compounds of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
Generally, the concentration of the compositions of the invention in a liquid composition, such as a lotion, will be from about 0.1-50 wt-%, preferably from about 0.5-5 wt %. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
The amount of the composition required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the attendant physician or clinician.
In general, however, a suitable dose will be in the range of from about 0.1 to about 150 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 1 to 100 mg/kg/day, most preferably in the range of 5 to 20 mg/kg/day.
The compositions are conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 xcexcM, preferably, about 1 to 50 xcexcM, most preferably, about 2 to about 30 xcexcM. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient(s).
The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
Following i.m. administration, the compositions of the invention enter the blood stream within about 10-15 minutes and reach a maximum concentration in the blood within one hour of administration, at which point they can be found throughout the circulatory related organs.